LOCUS       sequence-378919-        7150 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Fluorescent reporter for mitochondrial calcium.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7150)
  TITLE     Bacterial expression plasmid for production of mitochondrially 
            localized GCaMP6f
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 7150)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Dec 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: 
            "Unpublished"
COMMENT     Sequence Label: CMV-mt4xi-GCaMP6f
FEATURES             Location/Qualifiers
     source          1..7150
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(64..83)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        255..634
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        635..838
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     789..809
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     883..902
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        883..901
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             933..1007
                     /codon_start=1
                     /product="mitochondrial presequence of human cytochrome c 
                     oxidase subunit VIII (Rizutto et al., 1989)"
                     /label=COX8 presequence
                     /translation="MSVLTPLLLRGLTGSARRLPVPRAK"
     CDS             1038..1109
                     /codon_start=1
                     /product="mitochondrial presequence of human cytochrome c 
                     oxidase subunit VIII (Rizutto et al., 1989)"
                     /label=COX8 presequence
                     /translation="SVLTPLLLRGLTGSARRLPVPRAK"
     CDS             1140..1211
                     /codon_start=1
                     /product="mitochondrial presequence of human cytochrome c 
                     oxidase subunit VIII (Rizutto et al., 1989)"
                     /label=COX8 presequence
                     /translation="SVLTPLLLRGLTGSARRLPVPRAK"
     CDS             1239..1310
                     /codon_start=1
                     /product="mitochondrial presequence of human cytochrome c 
                     oxidase subunit VIII (Rizutto et al., 1989)"
                     /label=COX8 presequence
                     /translation="SVLTPLLLRGLTGSARRLPVPRAK"
     CDS             1374..1391
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             join(1431..1439,1440..1454)
                     /codon_start=1
                     /product="Xpress(TM) epitope tag, including an enterokinase
                     recognition and cleavage site"
                     /label=Xpress(TM) tag
                     /translation="DLYDDDDK"
     CDS             1482..1538
                     /codon_start=1
                     /product="calmodulin-binding peptide from avian smooth 
                     muscle myosin light chain kinase"
                     /label=calmodulin-binding peptide
                     /translation="RRKWNKTGHAVRAIGRLSS"
     primer_bind     complement(2139..2158)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     complement(2761..2778)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2767..2991
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      3037..3465
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3124..3143)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3334..3355
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3474..3494)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        3479..3808
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3659..3794
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3721..3740
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             3875..4669
                     /codon_start=1
                     /gene="aph(3')-II (or nptII)"
                     /product="aminoglycoside phosphotransferase from Tn5"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(3929..3948)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4539..4558
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    4843..4964
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4880..4899)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4934..4953
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5013..5029)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5013..5029)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5026..5048)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5037..5053
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     promoter        complement(join(5061..5067,5068..5085,5086..5091))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5106..5127
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5244..5261)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5415..6003)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5495..5514)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6174..6965,6966..7034))
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     6797..6816
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7035..7139)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gaaaagtgcc acctgacgtc gacggatcgg gagatctccc gatcccctat ggtgcactct
       61 cagtacaatc tgctctgatg ccgcatagtt aagccagtat ctgctccctg cttgtgtgtt
      121 ggaggtcgct gagtagtgcg cgagcaaaat ttaagctaca acaaggcaag gcttgaccga
      181 caattgcatg aagaatctgc ttagggttag gcgttttgcg ctgcttcgcg atgtacgggc
      241 cagatatacg cgttgacatt gattattgac tagttattaa tagtaatcaa ttacggggtc
      301 attagttcat agcccatata tggagttccg cgttacataa cttacggtaa atggcccgcc
      361 tggctgaccg cccaacgacc cccgcccatt gacgtcaata atgacgtatg ttcccatagt
      421 aacgccaata gggactttcc attgacgtca atgggtggag tatttacggt aaactgccca
      481 cttggcagta catcaagtgt atcatatgcc aagtacgccc cctattgacg tcaatgacgg
      541 taaatggccc gcctggcatt atgcccagta catgacctta tgggactttc ctacttggca
      601 gtacatctac gtattagtca tcgctattac catggtgatg cggttttggc agtacatcaa
      661 tgggcgtgga tagcggtttg actcacgggg atttccaagt ctccacccca ttgacgtcaa
      721 tgggagtttg ttttggcacc aaaatcaacg ggactttcca aaatgtcgta acaactccgc
      781 cccattgacg caaatgggcg gtaggcgtgt acggtgggag gtctatataa gcagagctct
      841 ctggctaact agagaaccca ctgcttactg gcttatcgaa attaatacga ctcactatag
      901 ggagacccaa gctggctagc ggatccgcca ccatgtctgt gctgacacct ctgctgctga
      961 gaggcctgac aggctctgct agaaggctgc ctgtgcctag ggccaagatc cactctctgc
     1021 cacctgaagg accttggagc gtgctgactc cactgctctt gaggggactc actggatctg
     1081 ctcggagact gccagtgcca cgcgctaaga ttcatagctt gccgcctgaa ggcggctcca
     1141 gtgttctgac accactgttg ctcaggggat tgactggcag cgcacgtaga ctccctgtgc
     1201 caagagccaa aattcacagt ctcccaccgg aaggcggttc cgtcctgaca cctttgttgt
     1261 tgcgcggctt gactggctca gcaagacggc tgccagttcc aagagcaaag atccacagcc
     1321 tgcctccaga gggccctaga tctggaagcg ctaaagaccc taccatgggt agccaccacc
     1381 accatcacca cggcatggct tctatgacag gcggccagca gatgggccgc gacctgtacg
     1441 atgacgacga taaggacctg gccacaatgg tggacagcag caggcggaag tggaacaaga
     1501 caggccacgc cgttagagcc atcggcagac tgtctagcct ggaaaacgtg tacatcaagg
     1561 ccgacaagca gaagaacggc atcaaggcta acttcaagat ccggcacaac atcgaggacg
     1621 gcggagtgca gctggcttac cactaccagc agaacacccc tatcggcgac ggaccagttc
     1681 tgctgcctga caaccactac ctgagcgtgc agagcaagct gagcaaggac cccaacgaga
     1741 agagggacca catggtgctg ctggaatttg tgaccgccgc tggcatcaca ctcggcatgg
     1801 acgaactgta caaaggcggc acaggcggct ctatggtgtc caagggcgaa gaactgttca
     1861 ccggcgtggt gcccattctg gtggaactgg atggggatgt gaacggccac aagttcagcg
     1921 tcagcggaga aggcgaaggc gacgccacat acggaaagct gaccctgaag ttcatctgca
     1981 ccaccggcaa actgcccgtg ccttggccta cactggtcac cacactgaca tacggcgtgc
     2041 agtgcttcag cagatacccc gaccatatga agcagcacga cttcttcaag agcgccatgc
     2101 ctgagggcta catccaagag agaaccatct tctttaagga cgacggcaac tacaagacca
     2161 gggccgaagt gaagttcgag ggcgacaccc tggtcaacag aatcgagctg aagggcatcg
     2221 acttcaaaga ggacggcaac atcctgggcc acaagctcga gtacaacctg cctgaccagc
     2281 tgaccgagga acagatcgcc gagttcaaag aagagttcag cctgttcgac aaggacggcg
     2341 acggcacaat caccaccaaa gaactgggca ccgtgatgag aagcctggga cagaacccta
     2401 cagaggccga gctgcaggac atgatcaacg aggttgacgc tgatggcgac ggaactatcg
     2461 acttccccga gttcctgacc atgatggcca gaaagatgaa gtacagggac acagaggaag
     2521 agatccgcga ggccttcggc gtgttcgata aggatggaaa cggctacatc tctgccgctg
     2581 agctgagaca cgtgatgacc aacctgggcg agaagctgac agacgaagag gtggacgaga
     2641 tgatcagaga ggccgacatt gacggcgacg ggcaagtgaa ctacgaagag tttgtccaga
     2701 tgatgaccgc caagtgaacc ggtgtcgacg aattcgggcc cgtttaaacc cgctgatcag
     2761 cctcgactgt gccttctagt tgccagccat ctgttgtttg cccctccccc gtgccttcct
     2821 tgaccctgga aggtgccact cccactgtcc tttcctaata aaatgaggaa attgcatcgc
     2881 attgtctgag taggtgtcat tctattctgg ggggtggggt ggggcaggac agcaaggggg
     2941 aggattggga agacaatagc aggcatgctg gggatgcggt gggctctatg gcttctgagg
     3001 cggaaagaac cagctggggc tctagggggt atccccacgc gccctgtagc ggcgcattaa
     3061 gcgcggcggg tgtggtggtt acgcgcagcg tgaccgctac acttgccagc gccctagcgc
     3121 ccgctccttt cgctttcttc ccttcctttc tcgccacgtt cgccggcttt ccccgtcaag
     3181 ctctaaatcg ggggctccct ttagggttcc gatttagtgc tttacggcac ctcgacccca
     3241 aaaaacttga ttagggtgat ggttcacgta gtgggccatc gccctgatag acggtttttc
     3301 gccctttgac gttggagtcc acgttcttta atagtggact cttgttccaa actggaacaa
     3361 cactcaaccc tatctcggtc tattcttttg atttataagg gattttgccg atttcggcct
     3421 attggttaaa aaatgagctg atttaacaaa aatttaacgc gaattaattc tgtggaatgt
     3481 gtgtcagtta gggtgtggaa agtccccagg ctccccagca ggcagaagta tgcaaagcat
     3541 gcatctcaat tagtcagcaa ccaggtgtgg aaagtcccca ggctccccag caggcagaag
     3601 tatgcaaagc atgcatctca attagtcagc aaccatagtc ccgcccctaa ctccgcccat
     3661 cccgccccta actccgccca gttccgccca ttctccgccc catggctgac taattttttt
     3721 tatttatgca gaggccgagg ccgcctctgc ctctgagcta ttccagaagt agtgaggagg
     3781 cttttttgga ggcctaggct tttgcaaaaa gctcccggga gcttgtatat ccattttcgg
     3841 atctgatcaa gagacaggat gaggatcgtt tcgcatgatt gaacaagatg gattgcacgc
     3901 aggttctccg gccgcttggg tggagaggct attcggctat gactgggcac aacagacaat
     3961 cggctgctct gatgccgccg tgttccggct gtcagcgcag gggcgcccgg ttctttttgt
     4021 caagaccgac ctgtccggtg ccctgaatga actgcaggac gaggcagcgc ggctatcgtg
     4081 gctggccacg acgggcgttc cttgcgcagc tgtgctcgac gttgtcactg aagcgggaag
     4141 ggactggctg ctattgggcg aagtgccggg gcaggatctc ctgtcatctc accttgctcc
     4201 tgccgagaaa gtatccatca tggctgatgc aatgcggcgg ctgcatacgc ttgatccggc
     4261 tacctgccca ttcgaccacc aagcgaaaca tcgcatcgag cgagcacgta ctcggatgga
     4321 agccggtctt gtcgatcagg atgatctgga cgaagagcat caggggctcg cgccagccga
     4381 actgttcgcc aggctcaagg cgcgcatgcc cgacggcgag gatctcgtcg tgacccatgg
     4441 cgatgcctgc ttgccgaata tcatggtgga aaatggccgc ttttctggat tcatcgactg
     4501 tggccggctg ggtgtggcgg accgctatca ggacatagcg ttggctaccc gtgatattgc
     4561 tgaagagctt ggcggcgaat gggctgaccg cttcctcgtg ctttacggta tcgccgctcc
     4621 cgattcgcag cgcatcgcct tctatcgcct tcttgacgag ttcttctgag cgggactctg
     4681 gggttcgaaa tgaccgacca agcgacgccc aacctgccat cacgagattt cgattccacc
     4741 gccgccttct atgaaaggtt gggcttcgga atcgttttcc gggacgccgg ctggatgatc
     4801 ctccagcgcg gggatctcat gctggagttc ttcgcccacc ccaacttgtt tattgcagct
     4861 tataatggtt acaaataaag caatagcatc acaaatttca caaataaagc atttttttca
     4921 ctgcattcta gttgtggttt gtccaaactc atcaatgtat cttatcatgt ctgtataccg
     4981 tcgacctcta gctagagctt ggcgtaatca tggtcatagc tgtttcctgt gtgaaattgt
     5041 tatccgctca caattccaca caacatacga gccggaagca taaagtgtaa agcctggggt
     5101 gcctaatgag tgagctaact cacattaatt gcgttgcgct cactgcccgc tttccagtcg
     5161 ggaaacctgt cgtgccagct gcattaatga atcggccaac gcgcggggag aggcggtttg
     5221 cgtattgggc gctcttccgc ttcctcgctc actgactcgc tgcgctcggt cgttcggctg
     5281 cggcgagcgg tatcagctca ctcaaaggcg gtaatacggt tatccacaga atcaggggat
     5341 aacgcaggaa agaacatgtg agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc
     5401 gcgttgctgg cgtttttcca taggctccgc ccccctgacg agcatcacaa aaatcgacgc
     5461 tcaagtcaga ggtggcgaaa cccgacagga ctataaagat accaggcgtt tccccctgga
     5521 agctccctcg tgcgctctcc tgttccgacc ctgccgctta ccggatacct gtccgccttt
     5581 ctcccttcgg gaagcgtggc gctttctcat agctcacgct gtaggtatct cagttcggtg
     5641 taggtcgttc gctccaagct gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc
     5701 gccttatccg gtaactatcg tcttgagtcc aacccggtaa gacacgactt atcgccactg
     5761 gcagcagcca ctggtaacag gattagcaga gcgaggtatg taggcggtgc tacagagttc
     5821 ttgaagtggt ggcctaacta cggctacact agaagaacag tatttggtat ctgcgctctg
     5881 ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt gatccggcaa acaaaccacc
     5941 gctggtagcg gtggtttttt tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct
     6001 caagaagatc ctttgatctt ttctacgggg tctgacgctc agtggaacga aaactcacgt
     6061 taagggattt tggtcatgag attatcaaaa aggatcttca cctagatcct tttaaattaa
     6121 aaatgaagtt ttaaatcaat ctaaagtata tatgagtaaa cttggtctga cagttaccaa
     6181 tgcttaatca gtgaggcacc tatctcagcg atctgtctat ttcgttcatc catagttgcc
     6241 tgactccccg tcgtgtagat aactacgata cgggagggct taccatctgg ccccagtgct
     6301 gcaatgatac cgcgagaccc acgctcaccg gctccagatt tatcagcaat aaaccagcca
     6361 gccggaaggg ccgagcgcag aagtggtcct gcaactttat ccgcctccat ccagtctatt
     6421 aattgttgcc gggaagctag agtaagtagt tcgccagtta atagtttgcg caacgttgtt
     6481 gccattgcta caggcatcgt ggtgtcacgc tcgtcgtttg gtatggcttc attcagctcc
     6541 ggttcccaac gatcaaggcg agttacatga tcccccatgt tgtgcaaaaa agcggttagc
     6601 tccttcggtc ctccgatcgt tgtcagaagt aagttggccg cagtgttatc actcatggtt
     6661 atggcagcac tgcataattc tcttactgtc atgccatccg taagatgctt ttctgtgact
     6721 ggtgagtact caaccaagtc attctgagaa tagtgtatgc ggcgaccgag ttgctcttgc
     6781 ccggcgtcaa tacgggataa taccgcgcca catagcagaa ctttaaaagt gctcatcatt
     6841 ggaaaacgtt cttcggggcg aaaactctca aggatcttac cgctgttgag atccagttcg
     6901 atgtaaccca ctcgtgcacc caactgatct tcagcatctt ttactttcac cagcgtttct
     6961 gggtgagcaa aaacaggaag gcaaaatgcc gcaaaaaagg gaataagggc gacacggaaa
     7021 tgttgaatac tcatactctt cctttttcaa tattattgaa gcatttatca gggttattgt
     7081 ctcatgagcg gatacatatt tgaatgtatt tagaaaaata aacaaatagg ggttccgcgc
     7141 acatttcccc
//
